C-13 MAGIC-ANGLE-SPINNING NMR ANALYSIS AND QUANTUM-CHEMICAL MODELING OF THE BATHOCHROMIC SHIFT OF ASTAXANTHIN IN ALPHA-CRUSTACYANIN, THE BLUE CAROTENOPROTEIN COMPLEX IN THE CARAPACE OF THE LOBSTER HOMARUS-GAMMARUS

Selective isotope enrichment, C-13 magic angle spinning (MAS) NMR, and semiempirical quantum chemical modeling, have been used to analyze li gand-protein interactions associated with the bathochromic shift of as taxanthin in alpha-crustacyanin, the blue carotenoprotein complex from the carapace of the lobster Homarus gammarus. Spectra of alpha-crusta cyanin were obtained after reconstitution with astaxanthins labeled wi th C-13 at positions 4,4', 12,12', 13,13', or 20,20'. The data reveal substantial downfield shifts of 4.9 and 7.0 ppm at positions 12 and 12 ' in the complex, respectively. In contrast, at the 13 and 13' positio ns, small upfield shifts of 1.9 ppm were observed upon binding to the protein. These data are in line with previously obtained results for p ositions 14,14' (3.9 and 6.8 ppm downfield) and 15,15' (0.6 ppm upfiel d) and confirm the unequal perturbation of both halves after binding o f the chromophore. However, these results also show that the main pert urbation is of symmetrical origin, since the chemical shift difference s exhibit a similar pattern in both halves of the astaxanthin molecule . A small downfield shift of 2.4 ppm was detected for the 4 and 4' pos itions. Finally, the 20,20' methyl groups are shifted 0.4 ppm upfield by the protein. The full data set provides convincing evidence that ch arge polarization is of importance for the bathochromic shift. The NMR shifts are compared with calculated charge densities for astaxanthin subjected to variations in protonation states of the ring-functional g roups, as models of ligand-protein interactions. Taking into account t he color shift and other available optical data, the current model for the mechanisms of interaction with the protein was refined. The resul ts point toward a mechanism in which the astaxanthin is charged and su bject to strong electrostatic polarizations originating from both keto groups, mast likely a double protonation.